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High-affinity binding of interferon-gamma to a

basement membrane complex (matrigel).

H Lortat-Jacob, … , H K Kleinman, J A Grimaud

J Clin Invest.

1991;

87(3)

:878-883.

https://doi.org/10.1172/JCI115093

.

Recently it was demonstrated that growth factors are bound to the extracellular matrix, and

can regulate cell behavior. Using three different types of binding assays, we have examined

the interaction of interferon-gamma with a basement membrane produced by the

Engelbreth-Holm-Swarm tumor. Basement membrane was found to bind interferon-gamma

in both a time- and concentration-dependent manner. Equilibrium binding analysis revealed

a high-affinity site with a dissociation constant of 1.5 10(-9) M and a maximum binding

capacity of 1.6 10(9) sites/mm2 of basement membrane. Competition studies show that the

binding is inhibited by heparan sulfate, suggesting that basement membrane-heparan

sulfate proteoglycan could be the binding site. This interaction was clearly confirmed by

native polyacrylamide gel electrophoresis and dot-blot analysis with purified basement

membrane molecules. Furthermore, the carboxy-terminal part of the interferon-gamma

molecule contains an amino acid cluster, very closely related to a consensus sequence,

present in more than 20 proteins known to bind sulfated glycosaminoglycans such as

heparin. These data demonstrate a possible role of extracellular matrix components in

storing cytokines and in modulating the cellular response to such factors.

Research Article

(2)

High-Affinity Binding of Interferon-ey

to

a

Basement Membrane

Complex (Matrigel)

Hugues Lortat-Jacob, Hynda K. Kleinman,* and Jean-Alexis Grimaud

Centre National delaRechercheScientifique URA602,InstitutPasteur, Lyon,France;and*Laboratory ofDevelopmentalBiology

andAnomalies, National InstituteofDentalResearch, National InstitutesofHealth, Bethesda, Maryland20892

Abstract

Recently it was demonstrated that growth factors are bound to the extracellularmatrix,and can regulate cell behavior. Using threedifferenttypesof binding assays, we have examined the interaction of

interferon-t

with a basement membrane pro-ducedby the Engelbreth-Holm-Swarm tumor. Basement mem-brane wasfoundtobind interferon-y in both a time- and

con-centration-dependentmanner.Equilibrium binding analysis re-vealed ahigh-affinity site with a dissociation constant of 1.5

10-'

Mand amaximum binding capacity of 1.6

10'

sites/mm2 of basement membrane. Competition studies show that the bind-ing is inhibited by heparan sulfate, suggestbind-ing that basement membrane-heparan sulfate proteoglycan could be the binding

site.Thisinteractionwasclearlyconfirmedbynative polyacryl-amide gel electrophoresis and dot-blotanalysis with purified basement membrane molecules. Furthermore, the carboxy-ter-minal part of the

interferon-'y

moleculecontains an amino acid cluster, very closely relatedto aconsensus sequence, present in morethan20 proteins known to bind sulfated

glycosaminogly-canssuch asheparin.These datademonstrate a possible role of extracellular matrix components in storing cytokines and in modulating the cellular response to such factors. (J. Clin.

In-vest. 1991.87:878-883.). Key words:bindingsite * extracellu-larmatrix * local concentration * heparan sulfate proteoglycan

Introduction

Increasing evidence nowdemonstrates that cellularactivities

depend on multifactorial events involvingsoluble-phase

fac-torssuchascytokines (1, 2),and/orsolid-phase factorssuch as extracellularmatrixcomponents(3,4). These soluble-or solid-phase factors bind to specific cell surface receptors, some of which have been characterized (5, 6). Basement membranes

arespecializedextracellularstructureswhich underlie epithelia

andarecomposed of collagen IV, laminin, nidogen/entactin,

and proteoglycans. Thesematricesaffectavariety of biological activitiesincludingcelladhesion(7,8),migration(8, 9), growth and differentiation, neurite outgrowth

(10),

regulation ofcell

morphology

(1

1),tumormetastases(6, 12-14), and regulation ofmorphogeneticprocessesduringdevelopment(15).The cel-lular response depends on the cell type and amount andform

ofthebasement membrane. Recently, it has been shown that allfourcomponents both alone and incombinationareableto exertbiological activity(8, 16-21). Laminin appears to be the

AddressreprintrequeststoDr.Grimaud,UnitedePathologie Cellu-laire, CNRS URA 602, Institut Pasteur, Avenue T.Garnier, 69365 Lyon Cedex 7,France.

Received.for

publication9May1990andinrevisedform14August

1990.

mostactive in this regard (10, 17, 20). The large heparan sulfate proteoglycan termed pearlecan has recently been cloned and

partially sequenced andcontainssome40%homology to the amino-terminal end of the laminin chains (21). It has been shown to promote hepatocyte adhesion and a specific receptor hasbeenisolated (22). This, however, does not appear to be the only function of the heparan sulfate which also servesto regu-late the passage of macromolecules through the basement membraneowing to its strongnegative charge (23).

Cytokines are soluble factors, with multiple overlapping cell regulatory actions (1). Theresponse ofa cell to a given

cytokineisdependent onthe local concentration of the cyto-kine, the cell type, and other cell regulators to whichit is

con-comitantly exposed (1).Forexample,

interferon-y

(IFN-"y)

isa

pleiotropic factorthat has manyeffectsondifferent cells (24).It

inducestheantiviralstate inuninfectedcells, can regulate cell growth and differentiation, regulates the immune response, and inhibits tumor cell growth (24-27). Although cytokines

suchas

IFN-'y

aregenerally thought ofassolublefactors, itis

possiblethattheseproteinsmay be inabound form, and thus, either be stabilizedtoprevent breakdownand/or immobilized to allow continued and localized stimulation ofcells.Recently, growth factorssuch as fibroblastgrowth factor (FGF),' have been found to be incorporated into the extracellular matrix where they are stored. In the caseof FGF, the thin extracellular

matrix ofthebasement membrane wasfoundtobeastorage depot and the major ligand washeparan sulfate

proteogly-can(28).

Here wehave investigatedwhether

IFN-"y

canbindto the basementmembrane. Since intactbasement membranefrom tissues is difficulttoisolatereproduciblyand inpurity,wehave usedareconstituedbasementmembrane from the

Engelbreth-Holm-Swarm(EHS)tumor tostudytheinteractionof IFN-y. This basementmembrane matrix, termed matrigel, contains

allofthe componentsof authenticbasementmembrane and in the electronmicroscope hassimilar lamina densa-like

seg-ments to those observed in tissue-derived basement

mem-branes(29-3 1).

Methods

1251I

iodinationof

IFN-"y.

Recombinant human

IFN-'y

withaspecific

activity of 2.10' U/mgwasa generousgift of Roussel Uclaf, Paris,

France. 10jgof IFN--ywasreacted in 50

Al

of 0.2Mphosphatebuffer,

pH 7.4,containing 0.8MNaCl,0.02% Tween20,500 MCi ofNa125I

(NewEngland Nuclear-DuPont, Boston, MA),and2.5jigof

chlora-mineT.AfterIminatambienttemperature,the reactionwasstopped

by addition of 10

AI

of sodium metabisulfiteat0.3 mg/ml. Proteins were thenseparated from free iodine by molecularsievingon a

pre-packedSephadexG25PD 10column(Pharmacia-LKB, Uppsala,

Sweden),equilibratedwith0.1Mphosphate buffer,pH 7.4, containing

The JournalofClinicalInvestigation,Inc.

Volume87, March 1991, 878-883

1.Abbreviations usedinthis paper: FGF, fibroblast growthfactor; GM-CSF, granulocyte/macrophagecolony-stimulatingfactor.

(3)

0.5 M NaCI, 0.02% Tween 20, and 10% of fetal calf serum. 251I-labeled

IFN-y,of which thespecificradioactivity was 20uCi/ug, was aliquoted and stored at-20°C. The high ionic strength buffer used here was foundtobe necessary topreventaggregation ofIFN--y.If labeled in more physiological conditions(e.g., PBS), IFN-'y eluted in the void volume of a G75 Sephadex column, as reported by others (32).

Preparationof matrigel-coated dish. Matrigel wasprepared as de-scribed (29). This mixture of matrix proteins is in solutionat4°C but forms a gel when warmedat24-37°C. Matrigel was thawedat4°C and diluted to 5 mg/ml with ice-cold 0.15MNaCl, 50mMTris-HCI, pH 7.4, and 30 ul per wellwasallowed to gel overnightat35°C ina96-flat botton well assay plate (Pro Bind 3915, Becton, Dickinson & Co., Lin-coln Park, NJ). Wellswerethensaturated for 1 h at 37°C with 200

,l

of 50 mMTris-HCI, pH 7.4,containing0. 15MNaCl and 20mg/ml BSA.

Binding studies.Ingeneral, 100 LIof unlabeledIFN-y(12.5ng/ml

to5

gg/ml),

containing25nCiof radiolabeled IFN-yemployedas a

tracer,wasincubated inmatrigel-coated wells,at roomtemperature. After the time necessary to reach equilibrium (or at different times for kineticstudies),thesupernatant fractionwasremoved and thematrigel

was rapidly washed with 200 Ml of PBS/BSA 0. 1%/Tween 20 0.02%. Bound radioactivitywasthen removedbydissolvingthematrigel with

4 M NaOH. The supernatant fraction plus the wash and dissolved matrigelwerecounted inagamma counter(1260 Multigamma, Phar-macia-LKB) to determine free and bound radioactivity. Experiments were performed twice, with triplicates for each data point. The quantity ofIFN-'ybound in each wellwascalculated from thespecific radioactiv-ity ofIFN-,yin each concentration. Nonspecificbinding was deter-mined according to Chamness and McGuire (33) using a lim(B/F)

= 0.2. To estimate Kd and Br,,,, (maximum binding capacity), data were represented as specific bound ligand plotted against thelogarithm

of free ligand concentration, according to the equation

B.

=M,

*Kd

.

F/(

I +

Kd*

F),

inwhichB,andM,are"surface

concentra-tions" ofbound IFN-y and coated matrigel, respectively, as de-scribed (34).

Enzymaticdigestion of heparansulfate. Matrigelwaspreparedas

described above and incubatedovernightwith50 Ml ofCaCl2(IMuM),

Tris-HCl(50 mM)containingeither 10 U ofheparitinase (Miles Scien-tificDiv.,Naperville, IL)or2.5 units ofheparinase (SigmaChemical Co., St.Louis, MO).Afterwashing, 100,ul of'2511IFN--y(25nCi)was

incubated for4 hwith the matrigel. Boundradioactivitywas deter-mined. Nonspecific bindingwascalculated with a200M excessof cold IFN.

Competitionstudy. Matrigel-coatedwellswereincubated (4 hat roomtemperature) with 100Mlof radiolabeled IFN-y(25 nCi/ml),in the presence ofincreasingconcentrations ofcompetitors (heparinfrom Serva,Heidelberg, FRG; heparansulfate fromSigmaChemicalCo.;

andrabbit anti-humanIFN-yfromGenzyme Co.,Boston,MA).

Non-specific bindingwasdetermined with a200 Mexcess of cold IFN. Boundradioactivitywasthendeterminedasdescribed above and the percentage ofbindinginhibitionwascalculated.

Nativepolyacrylamidegel electrophoresis(PAGE).This electropho-retic systemwasdevelopedtomonitor interactions between proteins that have different isoelectricpoints.The 5%acrylamide/0.5%bis acryl-amidegel,in 50mMTris/40mMboric acid, pH 8.5,wasallowedto

polymerizeasusual undervacuuminahorizontal gel mold. The comb was inserted in the middle of the gel.

Samples consisted ofIFN-y

(5Mgg)

aloneorinmixture with different matrix componentssuchaslaminin (5gg,GibcoLaboratories, Grand Island, NY), collagen typeIV(5

Mg,

Gibco Laboratories), heparan sul-fate (2.5 ag),andmatrigel (12.5 Mg). Beforeelectrophoresis, samples

wereincubated4 hat4°C. Tank buffer was Tris/borate buffer, pH 8.5.

Running conditionswereconstant voltage (200 V) for I h at 4°C. Proteinswererevealedby Coomassie Blue R-250 staining as usual.

Dot blots.Adescribed procedurewasfollowed to prevent glycosami-noglycans loss (35). Briefly, I ML ofglycosaminoglycan (from I to 10 mg/ml in PBS) was appliedonto anitrocellulose sheet and air-dried. Thenitrocellulosewasthen fixed in5%paraformaldehyde + 0.5% ce-tylpyridinium chloride for 30min at roomtemperature and washed in

buffer. Purified basement membraneproteins (10

Mg)

wereapplied

ontonitrocellulosebysuctionasusual. Thenonoccupied bindingsites

wereblocked withPBS/BSA3% for 30 minat37°C.Thenitrocellulose

stripswereincubatedovernightat4°Cwithinterferon-y

(1I

MinPBS/

BSA 1%),washed inPBS,andincubated with rabbitanti-IFN-y fol-lowedbyperoxidase-labeledanti-rabbitIgG. Alternatively radiola-beledIFNwasused,andafterwashing,thenitrocellulosefilterswere

directlycounted.

Domainmapping with monoclonal antibodies. MAbsB22, B27,32,

and35 which define differentepitopesonthe IFN-y moleculeare

de-scribed elsewhere(36).Radiolabeled IFN--ywasallowedtoreact, inthe

presence of differentMAbs,withheparansulfate

(1I

g),usingthe dot-blot assay. Afterwashing,each nitrocellulosestripwascountedto

de-termine the boundradioactivity.

Interferonassay. Antiviralactivity wasdetermined with a micro-titer inhibition ofcytopathiceffect assay

against

vesicularstomatitis virus(VSV) on amonolayerof Wish cells. Cells(5 x104/well)were

grown in 100MlofDMEsupplementedwith 10%fetal calfserum on

plasticor on basement membrane(30

Mig/well).

Before cellculture,

basement membraneswereincubated 6hat4°CwithIFN-yand

exten-sivelywashed.Then,24 hafter the addition of thevirus,cell

viability

wasdetermined with the

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assayasdescribed

(37).

In

parallel experiments,

IFN-vy

wasusedtodetermine theexact

quantity

ofIFN

remaining

on thebasementmembrane

just

beforecellculture.

Results

Binding of

IFN-,y

towhole basement membrane. Whenadded

to a

layer

of

gelled

basement

membrane,

radiolabeledIFN-y bindsina

time-dependent

manner

(Fig.

1).

Atroom

tempera-ture,the

steady

stateisreached in 3.5 h. Under thesame

condi-tions, binding

ofIFNto

plastic

(noncoated wells)

wasfoundto

be nonexistent.

Scatchard

analysis

of

IFN--y

binding

to basement

mem-branewascarriedout

(Fig.

2

a).

The

biphasic

curveindicateda

nonspecific

binding

(horizontal

part ofthe

plot)

and a

single

classof

binding

sites.Thedatawerecorrectedfor

specific

bind-ing

asdescribed

(33),

and

plotted

withthe

semilogarithmic

rep-resentation

(38)

(Fig.

2

b).

From the

s-shape

curve,

representing

the

binding site,

the dissociation constant

Kd

= 1.5 nM was

determined. A

high affinity

between IFN-y and basement

membranewasdemonstrated.The numberof

binding

siteswas

calculatedat 1.6

109/mm2

ofcoatedbasementmembrane.

Binding

inhibition assay.Wenextstudied thenatureofthe

interactionbetween IFN-y and basement membrane

(Fig.

3).

IFN-y was incubatedon

matrigel

inthe presence ofdifferent

components, and the inhibition of

specific binding

was

mea-sured.NaCl

(3 M),

butnotTween 20

(0.5%),

inhibited 100% of

the

binding,

suggesting

that the type of interaction ismore

ionicthan

hydrophobic.

As a

control,

polyclonal antibody

05

t "o

z 0

0

O i

Figure 1.Effectof time

on thebinding ofIFN--y

0 @ tobasementmembrane.

Basement

membrane-coated(e)ornoncoated

went m9mbrene *---- wells (o) wereincubated

IC c-O at room temperature

with 1.25 ngof radiolabeled IFN-y.

Afterthetimeindicated,

(4)

I

0~~~~~

2

z

a

z

0

0 25 50 75

BOUND IFN(ng)

1 Bmax/2=0.62sng

b

_ , d = i.snM

0 0.3 3

FREE FN ( nM)

Figure 2. Effect ofIFN-yconcentration

onthebindingofIFN-,ytobasement membrane. 25nCi/mIof radiolabeled

IFN-'ywasincubated for 4 h at room temperature, with 150,ugof coated basement membranecomplexand with increasing concentrations of coldIFN-,y

/ (from 12.5 ng/ml to 5 ug/ml). Bound andfreeIFN-,ywerethen measuredas

described in Methods.(a)Scatchard plot of the raw binding data. (b) Semi-logarithmic plot of the data corrected

30 for nonsaturable binding, using 0.2as

thelimit of bound/free.

against IFN-y (diluted 1/300)wasfound to fully preventthe

specific binding. Glycosaminoglycans such asheparan sulfate orheparin (20 ug/ml) inhibited thebindingtothe same extent.

Binding ofIFN--y to basement membrane maytherefore be due to the componentheparansulfate proteoglycans.

NativePAGEanalysisofthe binding. Electrophoresisunder native conditions was carried out to further investigate this binding, (Fig. 4). In this system,

IFN-y,

ofwhich the pI=8.7, is

slightly positive andwillmigratetowardthe cathode (lane 1). Incontrast, BSA (pl=5.2)isnegatively charged and migrated toward the anode(lane 2). When

IFN-y

andBSA aremixed, theirmigration is delayed(becauseof opposites, charges which

mayresultin someinteraction),but eachprotein migratestoits

side(lane 3). Heparan sulfate glycosaminoglycan, which is highly negative, should migrate (lane6a)toward the anode but cannotbe visualized because it is notstained by Coomassie Blue. When

IFN-y

is electrophoresed in thepresenceof hep-aransulfate (lane 6b), it migratestowardthe anodeinstead of thecathode, demonstrating thatthere isa strong interaction between IFN-'y and heparan sulfate. 2.5 Mg of heparan sulfate

wasable to completely bind IFN-y (5 ,g) and to permit its

migration

totheglycosaminoglycan side.

The same experimentwas performed with laminin (lane

4a) and laminin plusIFN(lane

4b)

orcollagentype IV(lane

5a)andcollagen typeIV plus IFN (lane5b). In allcases,the migration of IFN-,y is notsignificatively affected by the pres-enceof either lamininorcollagen type IV, showingthatthere

are no interactions between these components. Whole

base-mentmembrane complex (matrigel) and matrigel plus

IFN-'y

were also tested with two different preparations of matrigel (lanes 7 and 8). Only a small part of theIFN-'ymigrates on its side(cathode side) confirming that matrigelbindsIFN

(com-parecathode side oflane 1 with cathode side oflane 7b and 8b). Ascontrol, ribonuclease A (Sigma Chemical Co.), which is also basic-pI = 8.9 (lane 9a)-and ribonuclease A plus heparan sulfate(lane 9b)weretested, and ribonuclease migrationwas

notaffected by the presence ofheparansulfate.Thus,the inter-feron-heparan sulfate interaction appears specific.

Cathode side

-1 2

3

4

5 6

a

b

a

b

a

b

7

a

b

8

9

a

b

a b

_

-"_

_

___-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...

NaCI(H) Twin 2o(VI) HP(pg/ml)

INHIBITORS

HS(pg/ml) Ab(dilution)

Figure3. Effect of various inhibitorsonthebindingof IFN-yto

basement membrane.

IFN-'y

wascoincubated withNaCl,Tween20, heparin(HP), heparan sulfate (HS),orpolyclonalantibody against

IFN--y diluted in PBS/BSA 1%(Ab), 4h at roomtemperature. Nonspecific binding was determinedastheboundradioactivity in the presence ofa200M excessof cold IFN-y.

U

Anode

side

..._.p.:AO

Figure4.Effect of isolatedproteinsof the basementmembrane,and whole basement membrane complex,ontheIFN-,ymigrationina

native PAGE systematpH=8.5. Lane1,IFN(5gg). Lane2, BSA

(5 Mg).Lane3,IFN(5Mg)+BSA(5 Mug).Lane 4a, laminin(5 ag);4b, laminin (5,gg)+IFN(5pg).Lane5a,collagentype IV(5ag);5b, collagen typeIV(5 Ag)+IFN(5ag).Lane6a,heparansulfate(2.5

Ag); 6b, heparansulfate(2.5 ug) +IFN(5 ag).Lane 7a and 8a, matrigel(12.5ag), 7b and8b, Matrigel (12.5 ug)+IFN(5ug).Lane 9a, ribonucleaseA(5ag), 9b,ribonucleaseA(5Ag)+heparansulfate

(2.5

ug).

a4

.

a

z

0 U 0.3

a2

10

N

z

I-5 rc

0 0

liv-w- CMgo

I

I

9

3!

IMIR

RI

U)IS!

RI

% % %

(5)

Enzymaticdegradation ofbasement membrane-glycosami-noglycan. Matrigel was incubated with either heparinase or heparitinase. Radiolabeled

IFN-y

wasallowed to react as de-scribed above with enzyme-treated matrigel orwith untreated matrigel (to determine the 100% of binding). Digestion with heparinase completely abolished the binding, while digestion with heparitinase reduced it to 50% only. These data suggest that IFN binds to heparan sulfate heparin-like sequences, but we cannot rule out the fact that heparinase digestion may be more complete than that of the heparitinase.

Binding ofIFN--y topurified basement membrane

compo-nents. In order to measure the direct binding ofIFN-'yto puri-fied basement membrane components, ligand dot-blot experi-ments wereperformed. As shown in Fig. 5, only heparan

sul-fatebindsIFN-,y, confirming the binding activityfound inthe Scatchardanalysis. Whole basement membranecomplex(10

gg)

dot-blottedontonitrocellulose filters also bindsIFN-Y,but

weakly, presumably becauseof the lowcontentof heparan sul-fate(30).Theexperimentwasalso carriedoutwith other

extra-cellularmatrix components including collagen type I, III, and fibronectin. Noneofthese components boundIFN-y(data not shown).

In order to assess the specificity between heparan sulfate andIFN-y, radiolabeledIFNwas also allowed tointeractwith different glycosaminoglycans. As shown in Table I, keratan

sulfate,dermatansulfate, and chondroitin sulfatedo not

signifi-cantly bindIFN-y.Thisbindingseems tobe restricted to

hepa-rinand to heparan sulfate(the latter containing heparin-like sequences). The exactpolysaccharide residues involved in the

bindingremaintobedetermined.

Domainmapping. IFN-'ywasallowedto reactwith heparan

sulfate, in the presence ofMAbsto IFN--y. Among the four

MAbs tested, only B22 prevent thebindingbetween IFNand heparan sulfate (Table II). This MAb recognizes the last 15

amino-acids (carboxy-terminal side) ofthe

IFN-'y

molecule, whereas B27, 32, and 35 define other domains (36). These data demonstrate that thecarboxy-terminal partofIFN-,y is in-volved in its binding to heparan sulfate.

Antiviralactivity

of

basement membrane-bound interferon.

Basement membrane-coated wells were incubated with various

concentrations of

IFN-'y,

washed,and testedfor antiviral activ-ity.Asshown(Table III), cells grown on basement membrane-bound IFNwereprotected against thecytopathiceffect of the

virusin a dose-dependent manner, whereas cells grown on base-mentmembrane alone were not. Thus, the IFN--y bound to the basementmembrane isbiologically active.

*

_a

Figure 5. Dot-blot analysis of the binding of

IFN--y.IFN--y(1 MM)was incubated with

_.. cb

nitrocellulose

strips

dot-blottedwith 10 Ag of(a) heparan sulfate,(b)collagen type IV, (c) laminin, and(d)whole basement membranecomplex. After washing,IFN--y

_ wasstained with rabbit anti-humanIFN--y

-* U followedby peroxidaselabeledanti-rabbit IgG.

Table L. Interactions ofIFN-yandGlycosaminoglycans

Glycosaminoglycandot-blotted Nitrocellulose bound onto nitrocellulose filter radioactivity

cpm

Keratansulfate 117.1

Dermatansulfate 1,254.0 Chondroitin sulfate 2,140.6 Heparansulfate 13,438.5

Heparin 28,224.2

Radiolabeled IFN-ywasincubatedovernightat40C withthe indi-catedglycosaminoglycans (1zg) dot-blottedontonitrocellulose. After washingin PBS, the nitrocellulose strips were counted.

Discussion

Previous datareported thatextracellular matrix components may interact with growth factors such as FGF (28) or with

cytokines suchasIL-3or granulocyte/macrophage colony-stimulating factor (GM-CSF) (39). We present, for the first

time, the binding of human interferon--y to basement

mem-brane, asdetermined in several differentand specific binding

assays. Bindingdatarevealahigh-affinitysite forIFN--ywitha

Kdof1.5 x 10-9 M anda maximum binding capacity of 1.6

x

10-'

IFN

molecules/mm2

of basement membrane.

A relatively long time is needed for IFN-y binding. This couldbeexplained by the three-dimensionalstructure of the

basementmembranewhich could slow diffusion of molecules. Itshould be also noted that the heparansulfateproteoglycan

represents < 5% ofthe total protein present in matrigel (30). Thisalsoexplainstheimportance of nonspecific binding(B/F

= 0.2 in the Scatchard plot) which corresponds to IFN--y trapped, but not bound, in the basement membrane layers.

Thenumberofbasement membranebinding sitesis very

high

compared toIFN--y cellular receptor number(from 100to

10,000percell). This is well inexcessofthe numberof mole-cules neededto saturatecellularIFN--yreceptors(40). Further-more, theaffinity of IFN--y forbasement membrane is closeto

thereported

affinity

of this moleculefor its cellular receptor:

10-9 to

10-"

M (40).

Therefore,

we speculate that in vivo,

basement membrane likely interacts with this

cytokine

and thatthiscytokinecould be inanactivestate.

Inhibition experiments,

performed

to

identify

basement membrane components involved in IFN-y

binding,

showed

TableII. DomainMappingofIFN--y

MAb Boundradioactivity

100 B27 92.1 B22 14.6

32 87.5

35 112.1

Radiolabeled IFN--y wasincubated overnightat4°Cwith heparan sulfate (I ,ug) dot-blottedontonitrocellulose,in thepresence ofthe

(6)

TableIII. Basement Membrane-bound IFN Antiviral Activity

Basement membrane-bound IFN Viable cells

ng %

0.379 64.0

0.193 57.7

0.055 38.8

0.016 13.0

0.004 11.3

0.001 5.7

0 5.1

Wish cells (5 *104/well)wereculturedovernightonmatrigel preincu-batedwithvarious quantitiesof

IFN-'y,

and thenchallenged for24 h

withvesicularstomatitis virus. Cell viability was determined using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Matrigel-boundIFN wasestimated in parallelexperimentswith radiolabeled IFN.

that heparan sulfatewas aneffective competitor.

Electropho-reticand dot-blotanalysisconfirmedthespecificity ofthe bind-ing.Heparansulfateproteoglycanisthebinding site for IFN-'y,

asin thecaseof basic FGF,

IL-3,

andGM-CSF

(28,

39).

This is

also supportedby the fact thatheparinaseor

heparitinase

diges-tion of basement

membrane-glycosaminoglycan

significantly

reduced thebinding of

IFN-y.

The affinity of IFN-,y for heparan sulfate proteoglycan found here, 1.5 nM, ishigher thanthe

affinity

of basicFGF,

610 nM(28), although basicFGF(pl=9.8) ismorebasicthan

IFN-y

(pl = 8.7). This suggests that thebinding ofIFN to

basement membraneisnotbasedon asimple electrostatic

in-teraction,

but may involveadefined region ofthemolecule. Moreover, heparan

sulfate

cannot

significantly

fix

ribonucle-ase A (another basicprotein pl = 8.9) in our electrophoretic

system, showingthatthepresence ofa netpositive chargeon

the molecule isnot sufficient for

binding.

On the other side,

IFN--y

doesnot

bind

toothersulfated glycosaminoglycans such

asdermatan, keratan, orchondroitin sulfate, in spite of their negativecharge.Thus,this interaction appears to bespecific.

Thecarboxy-terminal partoftheIFN-ymolecule contains a clusterof nine amino acids:

Ala'24-Ser'32

(41) veryclosely relatedto a consensus sequence present in more than20known

heparin-binding proteins(42).Bindinginhibition experiments

have beendonewith fourMAbsdirectedagainst differentparts

of the IFN-y moleculetodetermine the

binding

siteonIFN-y. Only

antibodies

directed against the

carboxy-terminal

partof the moleculepreventthe

binding,

whereastheothers donot.

Our data and thatofotherinvestigators (28, 43)suggest that extracellularmatrixcomponents couldprovidealocal

concen-tration ofagivencytokine, directthe rangeof action ofsuch soluble

factors,

andactas a

physiological

storagedepotaround cells. In the caseof basic FGF, itwassuggestedthat the

regula-tionof capillarygrowth and the neovascular response may re-sult from displacement of growth factors from their storage siteswithinthebasement membrane and

surrounding

extracel-lularmatrix(28,44).Extracellularmatrix-heparan sulfatealso

bindsIL-3 and GM-CSF. Oncebound, these cytokinescan be presented in an activeformtohemopoieticcells,thus provid-ingamechanistic explanation forthe observed absolute depen-dence of haemopoietic cells on intimatestromal cell contact (43).Interferon-'y hasanextremely broad range of biological

activities on a great number of different cells (1, 24). Once

boundtobasement membranes,thismolecule is still active.

Acknowledgments

Weare grateful to Dr. J. P. Escande and "SANOFI-Recherche" for the grantobtained and encouragement in this research. Dr. J. Banchereau (Laboratory for Immunological Research, Schering-Plough) is also

gratefullyacknowledged for his kindgiftof MAbs.

This work was supported by afellowship (H. Lortat-Jacob) from the Societe d'HepatologieExperimentale.

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